Methods and primers for evaluating hiv-1 mutations

ABSTRACT

Primer sequences and a method of using such sequences for the genotyping of HIV-1-containing samples, particularly those which have failed genotyping analysis are provided using primer sequences designed for analysis of Group B subtype of the Group M type virus. For example, a combination of primers, including at least one species of forward primer and at least one species of reverse primer where the forward primer(s) can be represented by the degenerate sequence: RARRARGGGCTGYTGGARATGTS (Seq. ID No. 9) and the reverse primer(s) can be represented by the degenerate sequence: BCHTYACYTTRATCCCSGVRTARATYTGACT (Seq. ID No.: 10) or BCHTYACYTTRATCCCSGVRTARATYTGAC (Seq. ID No. 12) are suitably employed. The selected primers, one or more from each group, can be used as reverse transcription, amplification and sequencing primers and are suitably packaged in a genotyping kit. Such a kit may include reagents in addition to the primers, such as an RNase inhibitor, a reverse transcriptase, a polymerase, and/or dNTP and ddNTP feedstocks.

BACKGROUND OF THE INVENTION

The present application relates to methods and primers for evaluatingmutations in human immunodeficiency virus (HIV-1).

Human immunodeficiency virus is the primary causative agent of AcquiredImmune Deficiency Syndrome (AIDS), or AIDS-related complex (ARC). AIDSis an infectious disease characterized by generalized immunesuppression, multiple opportunistic infections, and neurologicaldisease. Although HIV is regarded to be the primary causative agent ofAIDS, multiple co-infecting clinical viral and bacterial pathogens areresponsible for the cluster of clinical syndromes seen in AIDS patients.

The clinical course of HIV infection is remarkable for its greatvariability. The clinical effects include increased susceptibility toopportunistic infections and rare cancers, such as Kaposi's sarcoma,neurological dysfunctions, leading to AIDS related dementias, andgeneralized immune dysfunctions.

The HIV-1 virus is a member of the lentivirus group of the retroviruses.Like all other retroviruses, it has an RNA genome which is replicatedvia the viral reverse transcriptase, into a DNA provirus which becomesintegrated into the host cell genome.

Various drugs are presently available to treat HIV. They fall into threedifferent classes—nucleoside reverse transcriptase inhibitors, or NRTI'ssuch as zidovudine, didanosine, zalcitabine, lamivudine, stavudine,abacavir, tenofovir, foscamet; non-nucleoside reverse transcriptaseinhibitors or NNRTI's such as nevirapine, delavirdine, efavirenz; andprotease inhibitors or PI's such as saquinavir, indinavir, ritonavir,nelfinavir, amprenavir, and lopinavir with ritonavir. Although some ofthese drugs may have similar modes of actions, resistance to one doesnot necessarily confer resistance to another.

Each of the presently available anti-retroviral compounds used to treatAIDS suffers from some disadvantages, including transient CD4 cell counteffects, incomplete inhibition of viral replication, toxicity atprescribing doses, and emergence of resistant forms of the virus. As aresult, combination therapies are being used to treat patients. Severalin vitro studies have suggested that the combination of two or moreanti-HIV compounds will more effectively inhibit HIV replication thaneach drug alone. Over the last several years, the standard of patientcare has evolved such that HIV patients are routinely treated withtriple drug combination therapy.

Combination therapy has significantly decreased HIV associated morbidityand mortality. However, a large number of patients are not able toachieve or maintain complete viral suppression even with combinationtherapy. Drug resistance is the consequence of this incomplete viralsuppression. The very high mutagenicity rate of HIV virus (due to theerror-prone nature of the viral reverse transcriptase) and the geneticvariability of the virus have led to many HIV variants with decreaseddrug susceptibility.

HIV-1 replication depends on a virally encoded enzyme, reversetranscriptase (RT) that copies the single-stranded viral RNA genome intoa double-stranded DNA/RNA hybrid. The HIV-1 RT enzyme lacks a 3′exonuclease activity which normally helps the “proof-reading” functionof a polymerase enzyme to repair errors. HIV-1 has a 9200-base genomeand, on average, RT makes at least one error during every transcriptionof 10,000 bases copied. Therefore, each progeny virus produced may beslightly different from its predecessor. The inaccuracy of RT results inan estimated in vivo forward mutation rate of 3×10⁻⁵ per baseincorporated. Mansky LM. Virology. 1996; 222:391-400.

Many mutations introduced into the HIV-1 genome will compromise theinfectivity of the virus; while some are compatible with virusinfectivity. The frequency with which genetic variants of HIV-1 aredetected in patients is a function of each variant's replicative vigor(fitness) and the nature of the selective pressures that may be actingon the population within the infected patient, Volberding PA, et al.,Antiretroviral therapy for HIV infection: promises and problems. JAMA.1998;279:1343-4. Selective pressures existing in HIV-1 infected personsinclude anti-HIV-1 immune responses, the availability of host cells thatare susceptible to virus infection in different tissues, and the use ofantiretroviral drug treatments.

The mutagenicity of the virus represents a significant barrier totreatment of the disease. Moreover, the mutagenicity of the virus makestesting for genetic changes in the virus very difficult. Testing forchanges in DNA sequence can proceed via complete sequencing of a targetnucleic acid molecule, although many persons in the art believe thatsuch testing is too expensive to ever be routine.

Attention has been increasingly focused on failure to achieve ormaintain viral suppression. Several factors may contribute to drugfailure, including poor patient adherence to treatment regimen, drugpotency, pharmacokinetic issues (related to antiretroviral drugabsorption, metabolism, excretion, and drug-drug interactions) and drugresistance Vella S, et al.Aids. 1998; 12:S147-8.b. Although multiplecombinations of antiretroviral drugs may suppress HIV-1 below the levelof HIV-1 RNA detection, this does not mean that the virus is notreplicating in “sanctuary” compartments. A therapy regimen may decreaseHIV-1 RNA to below detectable levels, but within months the HIV-1 viralload may increase again. If HIV-1 is replicating, resistance to therapycan develop.

Because HIV-1 replication occurs rapidly, large numbers of virusvariants, including those that display diminished sensitivity toantiretroviral drugs, are generated. Mutations that confer resistance toantiretroviral drugs can be present in HIV-1 infected persons beforeantiretroviral therapy is initiated due to transmission from anindividual having had prior therapy or due to spontaneously arisingmutations. Once drug therapy is initiated, the pre-existing populationof drug-resistant viruses can rapidly predominate because of a selectiveadvantage. For drugs such as lamivudine or nevirapine (and otherNNRTIs), a single nucleotide change in the HIV-1 RT gene can confer 100-to 1,000-fold reductions in drug susceptibility (Schinazi RF, et al IntAntiviral News. 1997;5:129-42). In vivo antiretroviral activity of thesedrugs, when used alone, is largely lost within 4 weeks of startingtherapy due to the rapid outgrowth of drug-resistant variants, RichmanDD, et al. Nevirapine resistance mutations of human immunodeficiencyvirus type 1 selected during therapy. J Virol. 1994; 68:1660-6. Somemutations selected by antiretroviral drugs directly affect viral enzymesand cause resistance via decreased drug binding, whereas others haveindirect effects., Condra JH, et al. J Virol. 1996; 70:8270-6, andHarrigan PR, et al. J Virol. 1996; 70:5930-4. Treatment with differentantiretroviral drugs may select for HIV-1 variants that harbor the same,or related, mutations. Treatments may even select for the outgrowth ofHIV-1 variants that are resistant to drugs to which the patient has notyet been exposed (cross-resistance).

Mutations can be detected by a technique called “single strandedconformational polymorphism” (SSCP) described by Orita et al., Genomics5: 874-879 (1989), or by a modification thereof referred to asdideoxy-fingerprinting (“ddF”) described by Sarkar et al, Genomics 13:441-443 (1992). SSCP and ddF both evaluate the pattern of bands createdwhen DNA fragments are electrophoretically separated on a non-denaturingelectrophoresis gel. This pattern depends on a combination of the sizeof the fragments and the three-dimensional conformation of theundenatured fragments. Thus, the pattern can not be used for sequencing,because the theoretical spacing of the fragment bands is not equal.

Others have attempted to determine the genetic status of the virus byprobe-based analyses, in which the presence or absence of a specificviral mutation is determined by whether or not an inquiry probehybridizes to the viral nucleic acid under specific hybridizationconditions. For example, Stuyver et al. (PCT International PublicationNo. WO 99/67428) describe the use of nucleic acid probe panels in areverse hybridization assay, and Gingeras et al. describe the use ofprobes to detect pairs of mutations (PCT International Publication No.WO 92/16180). Such assays may suffer from several deficiencies,including being unable to detect new viral mutants, and may not besensitive enough to cope with the complexity of many mutations within aregion.

Other methods include the use of resistance test vectors to culture hostcells with virus derived from a patient. The vector may include anindicator gene, such that when a test amount of an anti-HIV drug isadded to the cell culture, in an attempt to measure the resistance ofthe cloned virus to the drug in the cell culture system. (Parkin et al,U.S. Pat. No. 5,837,464).

By far, the most direct information about the genetic composition of thevirus in a patient is to directly determine the sequence of the virus(genotyping). The positive clinical benefit of genotyping has beendemonstrated in controlled retrospective and prospective interventionbased studies such as the Genotypic Antiretroviral Resistance Testing(GART)(Baxter JD, et al. A randomized study of antiretroviral managementbased on plasma genotypic antiretroviral resistance testing in patientsfailing therapy. AIDS; 2000;14;F83-F93 and VIRADAPT studies, Durant J,et al. Drug-resistance genotyping in HIV-1 therapy: the VIRADAPTrandomised controlled trial, Lancet. 1999; 353:2195-9 and Lancet 1999September 25;354(9184): 1128. The greater reduction in viral load whenthe identification of mutations associated with resistance to specificantiretroviral drugs is used as an adjunct to standard of care intreated patients has demonstrated the clinical benefit of the adjunctiveuse of genotyping to guide therapeutic decisions.

One of the difficulties of genotyping is the inherent variability andheterogeneity of the virus. Viruses have been found to be serologicallydifferent on the basis of reactivity of the host immune system to thevirus, and on the basis of ELISAs, antibody dependent cellularcytotoxicity assays (ADCC's), and CD4 inactivation procedures. Theextensive serologic heterogeneity of the virus is also mirrored in thegenetic sequences of the virus. As a result, the HIV-1 virus has beencategorized into two genetic groups, based on phylogeneticreconstruction using the viral DNA sequences. Group O (outlier)represents a minority of the HIV-1, and is thought to originate in WestAfrica, perhaps in Cameroon.

The vast majority of HIV-1 sequences that are associated with clinicalAIDS are of the Group M (major) type. Within the M group, there arevarious subtypes (also referred to as clades), having differentgeographic distributions, as shown below. HIV-1 Group M SubtypePredominant geographical location A (including A1 and A2) Central AfricaB Europe, North and South America, Australia, and Asia C East and SouthAfrica, India D Central Africa E Southeast Asia (Thailand) F (includingF1 and F2) South America (Brazil) and Eastern Europe (Romania) G CentralAfrica, Russia, and Portugal H Central Africa and Taiwan I Cyprus JCentral Africa and Europe K N O

Each subtype differs from the others in amino acid composition by atleast 20% in the viral envelope region, and at least 15% in the viralgag region. Within each subtype, the differences in env can be up to10%, while the differences in gag can be Up to 8%. The viral reversetranscriptase and protease genes, the sites known to be associated withdrug resistance, are found on the viral pol transcript. It is estimatedthat there is only a 75% similarity in amino acids between subtypes forHIV-1 pol. The variability at the nucleic acid sequence level is evengreater.

Retroviruses have propensity to recombine with related retroviruses. Ifone cell is infected with multiple viruses, recombination events mayoccur, leading to recombinant subtypes that may then infect otherindividuals. In addition to the various subtypes known, circulatingrecombinant subtypes have been observed, such as A/E (Central Africa),A/G (West and Central Africa), A/B (Kalingrad), A/G/H/K (Cyprus/Greece)as well as D/F, and B/D recombinants.

To date, the majority of clinical research in North America and WesternEurope has been directed to the Group M subtype B, due to its relativeprevalence over the other Group M subtypes. However, as the AIDSepidemic has spread, non-B subtypes are appearing with increasingfrequency in North America and Europe. In some instances, for example,an initial infected person with a non-B infection may serve as theinfection focal point for a local group, such that in some NorthAmerican centers (which remain predominantly B subtype), there can beentire localized population groups infected with non-B subtypes. Forexample, Group O and Subtype G of Group M have recently been found inAIDS patients arriving in the United States from Africa.

SUMMARY OF THE INVENTION

The present invention provides primer sequences, and a method of usingsuch sequences for the genotyping of HIV-1-containing samples,particularly those which have failed genotyping analysis using primersequences designed for analysis of Group B subtype of the Group M typevirus. Thus, a first aspect of the present invention is a combination ofprimers, including at least one species of forward primer and at leastone species of reverse primer. The forward primer(s) can be representedby the degenerate sequence: RARRARGGGCTGYTGGARATGTS (Seq ID No. 9)

optionally with an additional G at either or both ends, where thenon-standard letters (those others than A, C, G and T) reflect choicesof bases in accordance with conventional nomenclature as outlined below.There are a total of 128 possible sequences represented by thissequence. Variations of these sequences may also be employed. Forexample, Seq. ID Nos. 11, 15 and6 show primers where one G is added.Similarly, the reverse primer(s) can be represented by the degeneratesequence: AGTCARATYTAYBCWGGGATYAARGTRADGV (Seq. ID No.: 10) orGTCARATYTAYBCWGGGATYAARGTRADGV (Seq. ID No. 12)In the former case, there are a total of 3456 possible primer specieswithin this definition. In the latter case, the degenerate sequence alsorepresents 3456 possible sequences, differing only in the initial A. Theselected primers, one or more from each group, can be used as reversetranscription, amplification and sequencing primers.

The primers are suitably packaged in a genotyping kit. Such a kit mayinclude reagents in addition to the primers, such as an RNase inhibitor,a reverse transcriptase, a polymerase, and/or dNTP and ddNTP feedstocks.

The primers are suitably employed in the method of the invention. Inaccordance with this method, a sample suspected of containing a non-BGroup M HIV-1 virus or a Group O HIV-1 virus is treated to recover viralRNA. The recovered viral RNA is reverse transcribed to DNA, which issequenced using the primers of the invention. The resulting sequenceinformation is used to establish the genotype of the tested virus, i.e.,to determine to which subtype the virus in the sample belongs. Themethod of the invention may be practiced in parallel with genotypingprocedures that are designed to evaluate B-subtype virus. Alternatively,the method of the invention is practiced on samples that have previouslybeen the subject of a failed genotyping attempt using genotypingprocedures that are designed to evaluate B-subtype virus.

DETAILED DESCRIPTION OF THE INVENTION

While the terminology used in this application is standard within theart, the following definitions of certain terms are provided to assureclarity.

The term “allele” as used herein means a specific version of anucleotide sequence at a polymorphic genetic locus.

The term “polymorphic site” as used herein means a given nucleotidelocation in a genetic locus which is variable within a population.

The term “gene” or “genetic locus” as used herein means a specificnucleotide sequence within a given genome.

The term “location” or “position” of a nucleotide in a genetic locusmeans the number assigned to the nucleotide in the gene, generally takenfrom the cDNA sequence of the genomic sequence of a gene.

The nucleotides adenosine, cytosine, guanine and thymine are representedby their one-letter codes A, C, G, and T respectively. Inrepresentations of degenerate primers, the symbol R refers to either Gor A, the symbol Y refers to either T/U or C, the symbol M refers toeither A or C, the symbol K refers to either G or T/U, the symbol Srefers to G or C, the symbol W refers to either A or T/U, the symbol Brefers to “not A”, the symbol D refers to “not C”, the symbol H refersto “not G”, the symbol V refers to “not T/U” and the symbol N refers toany nucleotide. In the specification and claims of this application, adegenerate primer refers to any or all of the combinations of basechoices and to either DNA or the corresponding RNA sequence (i.e., withT replaced by U). Thus, a degenerate primer may represent a singlespecies, or a mixture of two species which fall within the choices, or amixture of three choices which fall with the choices, and so on up to amixture containing all the possible combinations.

The tenn “oligonucleotide primer” as used herein defines a moleculecomprised of more than three deoxyribonucleotides or ribonucleotides.Its exact length will depend on many factors relating to the ultimatefunction and use of the oligonucleotide primer, including temperature,source of the primer and use of the method. The oligonucleotide primeris capable of acting as an initiation point for synthesis when placedunder conditions which induce synthesis of a primer extension productcomplementary to a nucleic acid strand. The conditions can include thepresence of nucleotides and an inducing agent such as a DNA polymeraseat a suitable temperature and pH. In the preferred embodiment, theprimer is a SS oligodeoxyribonucleotide of sufficient length to primethe synthesis of an extension product from a specific sequence in thepresence of an inducing agent. In the preferred embodiment, theoligonucleotide primers are at least 18 nucleotides long. Sensitivityand specificity of the oligonucleotide primers are determined by theprimer length and uniqueness of sequence within a given sample oftemplate nucleic acid. Primers which are too short, for example, mayshow non-specific binding to a wide variety of sequences.

A first aspect of the present invention is a primer combinationcomprising, in a single solution, at least one forward HIV-1 primerselected from among primers comprising a sequence as represented by thedegenerate sequence of Seq ID Nos. 9, for example Seq. ID. Nos. 11, 15or 16, and preferably from among primers with exactly these sequences,at least one reverse HIV-1 primer selected from among primers comprisinga sequence as represented by the degenerate sequences of Seq. ID Nos. 10or 12, for example Seq. ID. Nos. 13 or 14, and preferably from amongprimers with exactly these sequences. Where the primers are to be usedas sequencing primers, the forward primers or the reverse primers arelabeled with a detectable label. For most common sequencing instruments,a fluorescent label is desirable, although other labels types includingcolored, chromogenic, fluorogenic (including chemiluminescent) andradiolabels could also be employed. The primer combination may includeother reagents appropriate for reverse transcription, amplification orsequencing, and may, of course, include HIV-1 genetic material foranalysis.

Specific forward primers for use in the primer combinations of theinvention are: GGAAAAAGGGCTGTTGGAAATGYG (Seq. ID No. 1)GRARRARGGGCTGTTGGAAATGTGG (Seq. ID No. 2) GRARRARGGGCTGTTGGAAATGTG (Seq.ID No. 3)

including without limitation the following non-degenerate primersequences: GGAAAAAGGGCTGTTGGAAATGTGG (Seq. ID No. 17)GGAAAAAGGGCTGTTGGAAATGTG (Seq. ID No. 18) GGAAAAAGGGCTGTTGGAAATGTCG(Seq. ID No. 19) GGAAAAAGGGCTGTTGGAAATGTC (Seq. ID No. 11)GAAAAAGGGCTGTTGGAAATGTG (Seq. ID No. 15) GAAAAAGGGCTGTTGGAAATGCG. (Seq.ID No. 16)

Specific reverse primers for use in the primer combinations of theinvention are: AGTCAGATTTACCCAGGGATTAAAGTAAGGV (Seq. ID No. 4)AGTCAGATTTACCCAGGGATTAAGGTAAGGV (Seq. ID No. 5)AGTCAGATTTACCCAGGGATCAAAGTAAGGV (Seq. ID No. 6)GYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq. ID No. 7)AGYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq. ID No. 8)

including without limitation the following non-degenerate primersequences: GTCAGATTTACCCAGGGATTAAAGTAAGGC (Seq. ID No.: 13)GCCAGATTTACCCAGGGATTAAAGTAAGGC (Seq. ID No.: 14)

The primer combinations described above can be used in a method inaccordance with the invention for a sample suspected of containing anon-B Group M HIV-1 virus or a Group O HIV-1 virus to assess the subtypeand genotype of the virus. The method comprises the steps of treatingthe sample to recover viral RNA; reverse transcribing the recoveredviral RNA; sequencing the reverse transcription product; and using theresults of the sequencing step to establish the genotype of the testedvirus. In this method, either or both of the reverse transcription stepand the sequencing step are performed using a primer combination asdescribed above. The method of the invention can include the step ofperforming a parallel genotyping procedure that is designed to evaluateB-subtype virus. Alternatively, the method can be utilized with a samplethat has previously been the subject of a failed genotyping attemptusing genotyping procedures that are designed to evaluate B-subtypevirus. This alternative method provides the advantage of not performingneedless testing on a sample that proves to be the presently more commonB-subtype.

EXAMPLE 1

Degenerate mixtures of forward primers of the sequenceGGAAAAAGGGCTGTTGGAAATGYG (Seq. ID No. 1)

and reverse primers of the sequence GYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq.ID No. 7)

were used to salvage non-B subtypes in samples that could not begenotyped using other primers. The TRUGENE HIV-1 genotyping kit (VisibleGenetics Inc., Toronto, Canada) was used to determine the genotype ofcertain non-B subtypes of HIV-1 virus. RNA was extracted from patientplasma samples according to the package instructions in a TRUPREPExtraction Kit forviral RNA. 17 ul of RNA is added to the amplificationmaster mix. The master mix contains (per reaction) 0.2 ul of SEQ ID No.1 (30 pmole/ul), 0.4 ul of SEQ ID No. 7 (30 pmole/ul), 6.4 ul of water,1.75 ul of dNTP, 1.165 ul of DTT, and 0.58 ul of Rnase inhibitor. Thereactions are thermocycled using a Perkin-Elmer 9700 thermocycler usingthe following temperature/time cycle program: 20 cycles 15 cycles 90 C.50 C. 94 C. 94 C. 57 C. 72 C. 94 C. 60 C. 70 C. 70 C. 4 C. 2 60 2 30 301.5 30 30 2 7 holdAfter incubating the amplification master mix and the RNA together forfive minutes at 50 C, 4 ul of a second master mix is added and theRT-PCR cycle program is continued. The second master mix contains 10 ulof RT-PCR buffer, 5 ul of Rnase inhibitor, 1 ul of reverse transcriptaseenzyme (Superscript, Invitrogen Corporation), and 17.5 ul of DNApolymerase. After the PCR cycle program was completed, samples weresequenced according to the protocol in the TRUGENE HIV-1 package insert(Visible Genetics Inc.).

The following samples were successfully amplified and sequenced. Viralload numbers are provided as copies of viral RNA per milliliter ofpatient plasma. “Origin” refers to the residence of the patient at thetime the plasma was collected. Sample ID Subtype ORIGIN TYPE Viral Load 1 I-001 B USA co-culture unknown  2 I-002 A/G USA co-culture unknown  3I-003 F USA co-culture 7.5 × 10⁷  4 I-004 B USA co-culture Unknown  5I-005 Group O USA co-culture Unknown  6 I-006 E USA co-culture 6.4 × 10⁶ 7 I-007 E USA co-culture 2.6 × 10⁹  8 I-008 B USA co-culture Unknown  9I-009 E USA co-culture 4.4 × 10⁵ 10 I-010 E USA co-culture unknown 11I-011 E USA co-culture 1.1 × 10⁷ 12 I-012 C USA co-culture 6.9 × 10⁶ 13I-013 B USA co-culture Unknown 14 I-014 F USA co-culture Unknown 15I-015 F/B USA co-culture 2.1 × 10⁶ 16 I-016 B USA co-culture Unknown 17I-017 C USA co-culture 5.6 × 10⁵ 18 1747 A NIH Repos. co-culture 2.5 ×10⁴ 19 2386 D NIH Repos. co-culture   4 × 10⁴ 20 W1-1 A Europe patientplasma 2.1 × 10⁴ 21 W1-2 A Europe patient plasma 1.4 × 10⁵ 22 W1-3 CEurope patient plasma 2.1 × 10⁴ 23 W1-4 D Europe patient plasma 6.2 ×10⁴ 24 W1-5 C Europe patient plasma 9.4 × 10⁴ 25 W1-6 A Europe patientplasma 8.5 × 10⁴ 26 W1-7 D Europe patient plasma 5.6 × 10⁵ 27 W1-8 FEurope patient plasma 5.7 × 10³ 28 W1-9 C Europe patient plasma Unknown29 W1-10 G Europe patient plasma Unknown 30 W1-11 F Europe patientplasma 1.6 × 10⁵ 31 W1-12 G Europe patient plasma 5.6 × 10⁵ 32 W1-13 GEurope patient plasma 2.3 × 10³ 33 W2-1 A Europe patient plasma 2.2 ×10⁴ 34 W2-2 A Europe patient plasma 1.4 × 10⁵ 35 W2-3 C Europe patientplasma 2.1 × 10⁴ 36 13866 B Israel patient plasma 1.4 × 10⁴ 37 15089 BIsrael patient plasma 1.0 × 10³ 38 15214 A Israel patient plasma 2.0 ×10³ 39 15422 C Israel patient plasma 1.1 × 10⁶ 40 16100 C Israel patientplasma 7.2 × 10⁵ 41 16242 C Israel patient plasma 7.2 × 10⁵ 42 16360 CIsrael patient plasma 2.6 × 10⁵ 43 16361 C Israel patient plasma 2.2 ×10⁵ 44 NA A/G Russia patient plasma 2.0 × 10⁵ 45 NA C Africa patientplasma 6.3 × 10³ 46 NA B Israel patient plasma 7.6 × 10⁴ 47 NA CEthiopia patient plasma 9.2 × 10⁴ 48 NA C Ethiopia patient plasma 4.1 ×10⁵ 49 NA C Ethiopia patient plasma 2.5 × 10⁴ 50 NA unknown Argentinapatient plasma 7.3 × 10³ 51 NA unknown Argentina patient plasma 3.6 ×10⁴ 52 NA C Ethiopia patient plasma 7.3 × 10⁴ 53 NA unknown Argentinapatient plasma 4.8 × 10⁵ 54 NA C Ethiopia patient plasma 1.2 × 10⁵ 55 NAC Ethiopia patient plasma 2.7 × 10⁵ 56 NA C Ethiopia patient plasma 1.9× 10⁵ 57 NA C Ethiopia patient plasma 5.3 × 10⁴ 58 NA C Ethiopia patientplasma 1.6 × 10⁴ 59 NA C Ethiopia patient plasma 1.1 × 10⁵ 60 NA CEthiopia patient plasma 9.3 × 10⁴ 61 NA C Ethiopia patient plasma 3.8 ×10⁴ 62 NA C Ethiopia patient plasma 1.0 × 10³ 63 NA C Ethiopia patientplasma 2.8 × 10⁴ 64 NA C Ethiopia patient plasma 1.4 × 10⁴ 65 NA CEthiopia patient plasma 7.5 × 10⁵ 66 NA B Israel patient plasma 5.5 ×10⁵ 67 NA unknown Israel patient plasma 3.2 × 10⁴ 68 NA C Ethiopiapatient plasma unknown 69 NA B Europe patient plasma unknown 70 NA KIvory Coast patient plasma unknown

EXAMPLE 2

Oligonucleotide primers in accordance with the present invention weretested to determine what percentage of non-B subtypes could be genotypedusing samples that could not be genotyped using the TRUGENE HIV-1genotyping kit. 74% of those samples were successfully genotyped.Similarly, greater than 95% of all samples known to be non-B sampleswere successfully genotyped using the oligonucleotide primers of thepresent invention. Panels of non-B subtype viruses were generated, usingpLAI as a positive control. After a viral load for each sample wasobtained (Roche Amplicor or Organon Teknika NASBA), samples werediluted, using a serial dilution procedure, down to 25 copies perreverse transcriptase reaction. In each case, successful base callingand mutation detection was achieved.

EXAMPLE 3

Using the primers of the present invention, successful amplification andsequencing of HIV-1 viruses from both Group M (various subtypes andrecombinants) and Group O was achieved. Mean % HIV-1 Type Number non-B'sSucessfully or Subtype tested Amplified A (Group M) n = >7  >95% B(Group M) n = >200  >95% C (Group M) n = >29  >95% D (Group M) n = >3 >95% E (Group M) n = >5  >95% F (Group M) n = >25  >95% G (Group M) n =3  >95% K (Group M) n = 1   100% Group O n = 2   100% Group M n = 6  100% Recombinants

1. A primer combination comprising, in a single solution, at least oneforward HIV-1 primer selected from among primers comprising a sequencerepresented by the degenerate sequence RARRARGGGCTGYTGGARATGTS (Seq IDNo. 9)

and at least one reverse HIV-1 primer selected from among primerscomprising a sequence represented by the degenerate sequenceAGTCARATYTAYBCWGGGATYAARGTRADGV (Seq. ID No.: 10) orGTCARATYTAYBCWGGGATYAARGTRADGV. (Seq. ID No. 12)


2. The primer combination of claim 1, wherein at least one forwardprimer comprises a sequence selected from the group consisting of:GGAAAAAGGGCTGTTGGAAATGTGG (Seq. ID No.: 17) GGAAAAAGGGCTGTTGGAAATGTG(Seq. ID No.: 18) GGAAAAAGGGCTGTTGGAAATGTCG (Seq. ID No.: 19)GGAAAAAGGGCTGTTGGAAATGTC (Seq. ID No.: 11) GGAAAAAGGGCTGTTGGAAATGYG(Seq. ID No.: 1) GRARRARGGGCTGTTGGAAATGTGG (Seq. ID No.: 2)GRARRARGGGCTGTTGGAAATGTG (Seq. ID No.: 3) GAAAAAGGGCTGTTGGAAATGTG (Seq.ID No.: 15) GAAAAAGGGCTGTTGGAAATGCG. (Seq. ID No.: 16)


3. The primer combination of claim 1, wherein at least one reverseprimer comprises a sequence selected from the group consisting of:AGTCAGATTTACCCAGGGATTAAAGTAAGGV (Seq. ID No. 4)AGTCAGATTTACCCAGGGATTAAGGTAAGGV (Seq. ID No. 5)AGTCAGATTTACCCAGGGATCAAAGTAAGGV (Seq. ID No. 6)GYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq. ID No. 7)AGYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq. ID No. 8)GTCAGATTTACCCAGGGATTAAAGTAAGGC (Seq. ID No.: 13)GCCAGATTTACCCAGGGATTAAAGTAAGGC. (Seq. ID No.: 14)


4. The primer combination of claim 3, wherein at least one forwardprimer comprises a sequence selected from the group consisting of:GGAAAAAGGGCTGTTGGAAATGTGG (Seq. ID No.: 17) GGAAAAAGGGCTGTTGGAAATGTG(Seq. ID No.: 18) GGAAAAAGGGCTGTTGGAAATGTCG (Seq. ID No.: 19)GGAAAAAGGGCTGTTGGAAATGTC (Seq. ID No.: 11) GGAAAAAGGGCTGTTGGAAATGYG(Seq. ID No.: 1) GRARRARGGGCTGTTGGAAATGTGG (Seq. ID No.: 2)GRARRARGGGCTGTTGGAAATGTG (Seq. ID No.: 3) GAAAAAGGGCTGTTGGAAATGTG (Seq.ID No.: 15) GAAAAAGGGCTGTTGGAAATGCG. (Seq. ID No.: 16)


5. The primer combination of claim 4, wherein the forward and reverseprimers are members of a set of degenerate forward and reverse primers,and the primer combination includes at least two species of degenerateforward and at least two species of degenerate reverse primers.
 6. Theprimer combination of claim 5, wherein the forward and reverse primerscomprise sequences as set forth in Seq. ID Nos. 1 and 7, respectively.7. The primer combination of claim 5, wherein the forward primers aremembers of the set of degenerate primers comprising the sequence:GGAAAAAGGGCTGTTGGAAATGYG. (Seq. ID No.: 1)


8. The primer combination of claim 7, wherein the forward primers havesequences as set forth in Seq. ID Nos. 15 and
 16. 9. The primercombination of claim 7, wherein the reverse primers are members of theset of degenerate primers comprising the sequence:GYCAGATTTACCCAGGGATTAAAGTAAGGC. (Seq. ID No. 7)


10. The primer combination of claim 9, wherein the reverse primers havethe sequence as set forth in Seq. ID Nos. 13 and
 14. 11. The primercombination of claim 5, wherein the reverse primers are members of theset of degenerate primers comprising the sequence:GYCAGATTTACCCAGGGATTAAAGTAAGGC. (Seq. ID No. 7)


12. The primer combination of claim 11, wherein the reverse primers havethe sequence as set forth in Seq. ID Nos. 13 and
 14. 13. The primercombination of claim 1, wherein the forward primers or the reverseprimers are labeled with a detectable label.
 14. The primer combinationof claim 13, wherein the detectable label is a fluorescent label.
 15. Agenotyping kit comprising at least one forward HIV-1 primer selectedfrom among primers comprising a sequence represented by the degeneratesequence RARRARGGGCTGYTGGARATGTS (Seq ID No. 9)

and at least one reverse HIV-1 primer selected from among primerscomprising a sequence represented by the degenerate sequenceAGTCARATYTAYBCWGGGATYAARGTRADGV (Seq. ID No.: 10) orGTCARATYTAYBCWGGGATYAARGTRADGV. (Seq. ID No. 12)


16. The kit of claim 15, wherein at least one forward primer is selectedfrom the group consisting of: GGAAAAAGGGCTGTTGGAAATGTGG (Seq. ID No.:17) GGAAAAAGGGCTGTTGGAAATGTG (Seq. ID No.: 18) GGAAAAAGGGCTGTTGGAAATGTCG(Seq. ID No.: 19) GGAAAAAGGGCTGTTGGAAATGTC (Seq. ID No.: 11)GGAAAAAGGGCTGTTGGAAATGYG (Seq. ID No.: 1) GRARRARGGGCTGTTGGAAATGTGG(Seq. ID No.: 2) GRARRARGGGCTGTTGGAAATGTG (Seq. ID No.: 3)GAAAAAGGGCTGTTGGAAATGTG (Seq. ID No.: 15) GAAAAAGGGCTGTTGGAAATGCG. (Seq.ID No.: 16)


17. The kit of claim 15, wherein at least one reverse primer is selectedfrom the group consisting of: AGTCAGATTTACCCAGGGATTAAAGTAAGGV (Seq. IDNo. 4) AGTCAGATTTACCCAGGGATTAAGGTAAGGV (Seq. ID No. 5)AGTCAGATTTACCCAGGGATCAAAGTAAGGV (Seq. ID No. 6)GYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq. ID No. 7)AGYCAGATTTACCCAGGGATTAAAGTAAGGC (Seq. ID No. 8)GTCAGATTTACCCAGGGATTAAAGTAAGGC (Seq. ID No.: 13)GCCAGATTTACCCAGGGATTAAAGTAAGGC. (Seq. ID No.: 14)


18. The kit of claim 17, wherein at least one forward primer is selectedfrom the group consisting of: GGAAAAAGGGCTGTTGGAAATGTGG (Seq. ID No.:17) GGAAAAAGGGCTGTTGGAAATGTG (Seq. ID No.: 18) GGAAAAAGGGCTGTTGGAAATGTCG(Seq. ID No.: 19) GGAAAAAGGGCTGTTGGAAATGTC (Seq. ID No.: 11)GGAAAAAGGGCTGTTGGAAATGYG (Seq. ID No.: 1) GRARRARGGGCTGTTGGAAATGTGG(Seq. ID No.: 2) GRARRARGGGCTGTTGGAAATGTG (Seq. ID No.: 3)GAAAAAGGGCTGTTGGAAATGTG (Seq. ID No.: 15) GAAAAAGGGCTGTTGGAAATGCG. (Seq.ID No.: 16)


19. The kit of claim 15, wherein the forward and reverse primers aremembers of a set of degenerate forward and reverse primers, and theprimer combination includes at least two species of degenerate forwardand at least two species of degenerate reverse primers.
 20. The kit ofclaim 19, wherein the forward primers are members of the set ofdegenerate primers comprising the sequence: GGAAAAAGGGCTGTTGGAAATGYG.(Seq. ID No.: 1)


21. The kit of claim 20, wherein the forward primers have the sequenceas set forth in Seq. ID Nos. 15 and
 16. 22. The kit of claim 20, whereinthe reverse primers are members of the set of degenerate primerscomprising the sequence: GYCAGATTTACCCAGGGATTAAAGTAAGGC. (Seq. ID No. 7)


23. The kit of claim 22, wherein the forward primers have the sequenceas set forth in Seq. ID Nos. 15 and
 16. 24. The kit of claim 23, whereinthe reverse primers have the sequence as set forth in Seq. ID Nos. 13and
 14. 25. The kit of claim 19, wherein the reverse primers are membersof the set of degenerate primers comprising the sequence:GYCAGATTTACCCAGGGATTAAAGTAAGGC. (Seq. ID No. 7)


26. The kit of claim 25, wherein the reverse primers have the sequenceas set forth in Seq. ID Nos. 13 and
 14. 27. The kit of claim 15, whereinthe forward primers or the reverse primers are labeled with a detectablelabel.
 28. The kit of claim 27, wherein the detectable label is afluorescent label.
 29. The kit of claim 15, wherein the kit furthercomprises one or more reagents selected from the group consisting of anRNase inhibitor, a reverse transcriptase, a polymerase, and dNTP andddNTP feedstocks.
 30. The kit of claim 29, wherein the forward primersor the reverse primers are labeled with a detectable label.
 31. The kitof claim 30, wherein the detectable label is a fluorescent label.
 32. Amethod for evaluating a sample suspected of containing a non-B Group MHIV-1 virus or a Group O HIV-1 virus to assess the type of the virus,comprising the steps of: treating the sample to recover viral RNA;reverse transcribing the recovered viral RNA; sequencing the reversetranscription product; and using the results of the sequencing step toestablish the genotype of the tested virus, wherein at least one of thereverse transcription step and the sequencing step is performed using aprimer combination in accordance with claim
 1. 33. The method of claim32, further comprising the step of performing a parallel genotypingprocedures that is designed to evaluate B-subtype virus.
 34. The methodof claim 32, wherein the sample is one that has previously been thesubject of a failed genotyping attempt using genotyping procedures thatare designed to evaluate B-subtype virus.